With the development of the in vitro agar cloning technique for quantitating an apparent committed granulocytic stem cell pool, study of regulatory factors in the early stages of granulopoiesis is now possible. Factors have been discovered in the sera and urine of patients and mice with leukemia which apparently cause resting stem cells to enter the proliferative phase and give rise to partially differentiated granulocytic colonies in vitro. A factor found in normal urine appears to inhibit colony formation. With the use of mouse marrow cells, we have established two linear dose response assays to study stimulatory substances. One uses the in vitro agar cloning technique. The second measures tritiated thymidine incorporation into nucleic acids. Fractions obtained by column chromatography from human leukemic urine have been found to have divergent activities when assayed by the two methods. Our objectives are to isolate and purify these regulatory factors by employing standard procedures of protein purification involving chromatography, salt fractionation, electrofocusing and electrophoresis. We plan to study the effects of perturbations of granulopoiesis by chemotherapy and leukapheresis as a means of isolating the humoral regulators in normal and neoplastic diseases in animals and man to determine the physiologic role these substances play in health and disease. Furthermore, we plan to study the mechanism of action of these factors by studying the effect on the uptake of radioactive isotopes involved in protein RNA and DNA synthesis in intact cells and isolated chromatin. It is hoped that methods can be developed for studying the enzymatic steps involved in the action of these factors.